Organic Cations and Mitochondrial Efficiency Abstract Metformin, the most widely prescribed drug for type 2 diabetes, is also recognized for a variety of other beneficial effects including protection against ischemia/reperfusion injury in cardiac cells, anti-proliferation in cancer cells, attenuation of atherosclerosis and vascular senescence in the context of high fat diets, extension of lifespan in mice, and weight loss in overweight and obese patients. Despite over half a century of use, the primary mechanism of action of metformin has not been established. Using a nutrient sensitive cell-based strategy, Gohil et al. (Nature Biotechnology 28:249, 2010) recently screened ~3700 commercially available chemical compounds and identified more than 80 that interfere with energy production by the mitochondria, similar to metformin. Several of the most potent compounds were already known to directly inhibit mitochondria, but most had not been previously linked to energy metabolism. Of those remaining compounds, nearly all carry a positive charge at physiological pH, also similar to metformin. Any positively charged small molecular compound will enter cells and diffuse to the negatively charged mitochondrial matrix, provided the molecule is naturally membrane permeant or can enter via a transporter. Based on basic principles of electrochemical gradients (i.e., Nernst equilibria), the accumulation of a stable positively charged compound in the mitochondrial matrix will decrease the net proton motive force available to drive ATP synthesis, thereby decreasing the efficiency of aerobic energy production. The central hypothesis of this project is that organic cations decrease mitochondrial bioenergetic efficiency, and that the consequent increase in energy expenditure is the underlying mechanism by which such compounds improve glycemic control in the context of high fat diet-induced insulin resistance. Aim 1 will establish the impact of organic cations on mitochondrial and cellular bioenergetic function. Aim 2 will determine the biochemical mechanism(s) and mitigating properties by which organic cations alter bioenergetic function, and Aim 3 will determine if decreased mitochondrial efficiency is the underlying mechanism by which organic cations protect against high fat diet-induced obesity and insulin resistance. This project is expected to significantly advance our understanding of how mitochondrial bioenergetic function may be manipulated to prevent or treat diabetes, as well as other diseases associated with disorders of metabolism.